Sage Journals: Discover world-class research

Abstract

This study aimed to investigate the oxidative stress, hypoxia biomarkers, and circulating microparticles (MPs) in β thalassemia major. The study included 56 children with thalassemia and 46 healthy controls. Hypoxia biomarkers, oxidative stress biomarkers, and total plasma fragmented DNA (fDNA) were detected by the standard methods. The MPs were assessed by flow cytometry. Hypoxia and oxidative stress biomarkers, fDNA, and MPs were higher and total antioxidant capacity (TAC) was lower in patients with thalassemia than the controls. In splenectomized patients and those who had complications, vascular endothelial growth factor (VEGF), malondialdehyde, fDNA, endothelial, platelet, and activated platelet MP levels were higher while, TAC was lower than the nonsplenectomized patients. In conclusion, the increased tissue hypoxia, oxidative stress in β thalassemia, and its relationship with DNA damage and MPs release could explain many complications of thalassemia and may have therapeutic implications. The VEGF could serve as an important indicator for adequacy of blood transfusion in thalassemia.

Keywords

β thalassemia major fragmented DNA circulating microparticles oxidative stress hypoxia

Introduction

The profound anemia of homozygous β thalassemia and the high oxygen affinity of the blood produced combine to cause severe tissue hypoxia. Tissue hypoxia increases the expression of erythropoietin (EPO). In thalassemia, due to ineffective erythropoiesis, the imbalance between erythrocyte supply and demand persists despite increased tissue hypoxia and increased EPO. As a result, EPO level remains high and the marrow typically becomes hypercellular.^1,2 Over time, the combination of tissue hypoxia, increased EPO, and ineffective erythropoiesis creates a vicious cycle that may ultimately lead to a massive expansion of erythroblasts. Eventually, secondary bony pathologies³ and extramedullary erythropoiesis⁴ can also develop.

The hypoxia-inducible factor (HIF) pathway plays a protective role in regulating genes that mitigate the effects of low oxygen tension. Under normoxic conditions, oxygen-sensitive HIF-α isoforms are rendered inactive via proline hydroxylation by HIF-specific prolyl hydroxylases. Under hypoxic conditions, HIF-α isoforms are stabilized, heterodimerize with HIF-β, and translocate to the nucleus where they bind to hypoxia-responsive element motifs.^5
–7 In cooperation with other transcriptional coactivators, HIF induces transcription of genes that ameliorate the effects of hypoxia, including EPO and its receptor, transferrin and its receptor, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor (VEGF).⁸ The VEGF is a chemoattractant that enables the homing of regenerative endothelial cells into local vascular lesions.⁹ There is still a considerable debate over the vasculoprotective versus proinflammatory effects of VEGF.¹⁰

Oxidative stress in thalassemia primarily caused by the red blood cell (RBC) abnormalities, the oxidation of globin subunits in thalassemia erythroid cells, leads to the formation of hemichromes. Hemichromes bind to or modify various components of the mature RBC membrane, such as protein band, ankyrin, and spectrin.¹¹ After the precipitation of hemichromes, heme disintegrates and toxic nontransferrin-bound iron (NTBI) species are released. Other contributing factors to iron overload are increased intestinal absorption and regular blood transfusions. Consequently, free iron species, NTBI and labile plasma iron catalyze the formation of reactive oxygen species (ROS).¹² This oxidative stress may contribute to shortened life span of erythrocytes, primary or secondary amenorrhea, hypogonadism, osteoporosis, heart failure, endocrine abnormalities like diabetes, hypothyroidism, liver failure, and ultimately early death.¹³

Cell membrane microparticles (MPs) are phospholipid microvesicles shed from the plasma membrane of cells undergoing activation or apoptosis. The MPs are present in healthy individuals. However, an increase in their release is a controlled event and is considered a hallmark of cellular alteration. The MPs display cell surface proteins that indicate their cellular origin. In addition, they may also express other markers, for example, markers of cellular activation.¹³ Most MPs are highly procoagulant, expressing annexin V binding sites, and are capable of interacting with other cells to participate in various physiologic and pathologic processes, especially thrombosis and inflammation.^15,16 Oxidative stress is one of the risk factors involved in increasing the MPs formation.¹⁷ At the same time, oxidative stress is generated by MPs.¹⁸

To date there is no prior study investigating oxidative stress, hypoxia biomarkers, fDNA and circulating MPs together in children with thalassemia in Upper Egypt. We supposed that impaired response to tissue hypoxia, subsequent oxidative stress, and MPs release would be major determinants of the clinical outcome of patients with thalassemia. This study aimed to investigate the oxidative stress, hypoxia biomarkers, and circulating MPs in β thalassemia major.

Materials and Methods

Patients

A total of 56 pediatric patients with thalassemia were voluntarily recruited to this prospective cross-sectional controlled study in the Thalassemia Centre of Assiut Children University Hospital, Assiut, Egypt, during the period from January 2011 to September 2011. Investigations were compared to the levels in 46 socioeconomically , age- and sex-matching healthy control group of volunteers. Controls and patients were on a normal diet without vitamin supplementation and were free from any infection or medication. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki and informed consent was obtained in every case from their parents. Demographic, clinical, routine, and investigative biochemical characteristics of all participants were reported.

Inclusion Criteria

Patients with β-thalassemia who visit the Pediatric Hematology unit for follow-up and for blood transfusion. They were on irregular blood transfusion and chelation therapy with subcutaneous administration of desferrioxamine with variable compliance.

Exclusion Criteria

Patients on antioxidants or with known cardiac, infectious, inflammatory, or pulmonary diseases were excluded from the study.

Biochemical Investigations

Blood samples (5 mL) were collected from fasting and resting participants through venipuncture in pyrogen-free EDTA/sodium fluoride (3 mL) or plain tubes. Serum was separated and aliquot frozen at −20°C till used. Specific enzyme-linked immunosorbent assay (ELISA) was utilized to measure the plasma levels of each of VEGF (Cat# ELH-VEGF-001, RayBiotech, Inc, Norcross, Georgia) and HIF-1α (Cat# DYC1935-5, R&D Systems, Minneapolis, Minnesota) with specific capture/biotinylated detection antibodies and streptavidin–horseradish peroxidase/tetramethylbenzidine as the detection system. Serum total antioxidants capacity (TAC) and total peroxides (TPs; organic and inorganic) were colorimetrically measured according to the methods described by Koracevic et al¹⁹ and Harma et al.²⁰ Oxidative stress index (OSI) was the ratio of TP/TAC. Serum thiobarbituric acid (TBA) reactive substances and malondialdehyde (MDA) were measured in a reaction mix containing 50 μL of sample and 25 µL of 10% sodium dodecyl sulfate, mixed with 100 µL TBA (0.530 g/dL in 100 mmol/L sodium acetate buffer, pH 3.2) and 10 µL butylated hydroxytoluene (0.3 mmol/L in absolute ethanol). The mixture was incubated at 90°C for 1 hour, then cooled and centrifuged at 10 000 rpm for 10 minutes at 4°C. Optical density (OD) of the pink chromogen was measured against H₂O as blank at 540 nm. 1,1,3,3-Tetraethoxypropane (100 mmol/L).²¹ Plasma pyruvate was measured following the decrease in ultraviolet absorption by NADH/H⁺ according to instructions of the manufacturer (Cat# 180 000, Greiner Diagnostics GmbH, D-79353 Bahlingen, Germany). Plasma lactate was estimated colorimetrically according to instructions of the manufacturer (Cat# 274 001, Spectrum Biodiagnostics, Cairo, Egypt). The lactate/pyruvate (L/P) ratio was calculated. Plasma total fDNA was measured after precipitation with perchloric acid (20 µL 7 mol/L perchloric + 260 µL sample) for 10 minutes, then mixed and centrifuged at 10 000 rpm for 10 minutes at 4°C. One hundred fifty microliters of supernatant was mixed with 300 µL of diphenylamine (0.088 mol/L in 98%, v/v, glacial acetic acid: 1.5%, v/v, sulfuric acid: 0.5%, v/v, of 1.6% acetaldehyde), incubated at 50°C for 1 hour, and the OD of the purple chromogen was measured at 575 nm against H₂O as blank and using DNA as a standard.²² Determination of serum ferritin was done by ELISA kit manufactured by Diagnostic Biochemist Canada Inc (41 Byron Avenue, Dorchester, Ontario Canada) (cat. No. CAN – f – 4280 version 5.0).

Microparticles Isolation and Characterization

Blood samples were collected into a 5 mL tube containing 3.2% citrate. In order to isolate the MPs, cells were removed by centrifugation for 20 minutes at 1550g at 20°C within 15 minutes after collection. Then, 250 µL of plasma were centrifuged for 30 minutes at 18 800g at 20°C. After centrifugation, the supernatant was removed and the pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged for 30 minutes at 18 800g at 20°C. The supernatant was removed again and MPs pellet was resuspended in PBS.²³

Flow cytometric analysis was used to quantify and characterize MPs. Five microliters of MPs sample were diluted in 35 µL of PBS containing 2.5 mmol/L calcium chloride. The samples were then incubated for 20 minutes at room temperature in the dark with 5 µL of fluoroisothiocyanate-conjugated annexin V (IQ products, Netherland) and 5 μL of phycoerythrin and peridinium-chlorophyll-protein-conjugated cell-specific anti-human monoclonal antibody. After incubation, PBS/calcium buffer was added and the samples were analyzed on a fluorescence activated cell sorter (FACS) Caliber flow cytometry with Cell Quest software (Becton Dickinson Biosciences, San Jose, California, USA). Fifty thousand events were analyzed, and MPs were reported as a percentage of the total events. Anti-human immunoglobulin G was used as an isotype-matched negative control for each sample.^23,24

Microparticles were identified on the basis of their forward scatter compared to that of calibrate reference beads of 1.0 µm to calibrate the size range of MPs (latex beads, amine-modified polystyrene, fluorescent red aqueous suspension, 1.0 μm mean particle size, Sigma-Aldrich Chemie Gmbh Munich, Germany) and positivity for annexin V. The microparticles express phosphatidylserine, which is detected by annexin V labeling.²³ Microparticles subpopulations were identified by their ability to bind cell-specific monoclonal antibodies. Detection of endothelial MPs was performed using anti-CD146 labeling (Beckman coulter, France). Detection of platelet MPs was performed using CD41 and/or CD61 (Becton Dickinson Biosciences). Erythrocyte, monocytes, and leukocyte MPs were detected by anti-glycophorin A (Becton Dickinson Biosciences), CD14, and anti-CD45 (Beckman coulter), respectively. Anti-CD62P (Becton Dickinson Biosciences) labeling was used to detect P-selectin positive MPs (activated platelet MPs). ^25,26

For detection of MPs and MPs subpopulation, forward- and side-scatter histogram were used to define the MPs (R1) according to the size of the reference calibrate bead. Events defined as MPs (R1) were then selected for their annexin V binding, determined by positivity for annexin V (R2). Then annexin V positive MPs (R2) was further examined for expression of cell-specific antibodies such as CD 61, CD41, and glycophorin A (Figure 1). Total MPs are reported as percentage of total events. Annexin V positive was reported as the percentage of total MPs. The percentage of MPs subpopulation such as platelet, endothelial, and other are expressed as percentage of annexin V positive population.

Figure 1.

Flow cytometric analysis of microparticles: a representative set of scattergrams of a sample from a patient with thalassemia showing microparticles (MPs) and MPs subpopulations. Forward and side scatter histogram was used to define the MPs (R1) according to the size of reference calibrate bead (A). Events defined as MPs (R1) were then selected for their annexin V binding, determined by positivity for annexin V (R2) (B). Then annexin V positive MPs (R2) were further examined for expression of cell-specific antibodies such as CD 61, CD41, and anti glycophorin A (C, D) compared to the negative isotype control (not shown).

Statistical Analysis

Data analysis was done using statistical package for social sciences (SPSS), version 16. Data were expressed as the mean ± standard error of mean. Results were analyzed statistically using Mann-Whitney U analysis. Correlation among the investigated parameters in each group was tested by the nonparametric Spearman analysis. A P value ≤.05 denoted the presence of a statistically significant difference.

Results

The most relevant characteristics of patients are shown in Table 1. Differences between healthy controls and pediatric patients with thalassemia in the levels of the investigated hypoxia and oxidative stress biomarkers are shown in Table 2. There was a significantly higher fDNA, hypoxia biomarkers (lactate, L/P ratio, VEGF, HIF-1α), and oxidative stress markers (OSI, MDA) and significantly lower TAC in patients with thalassemia than the healthy controls. The differences in some clinical characteristics and the investigated hypoxia and oxidative stress biomarkers between the splenectomized patients and nonsplenectomized patients with thalassemia are shown in Table 3. There was a significantly lower TAC and significantly higher VEGF, MDA, and fDNA levels in splenectomized than in nonsplenectomized patients with thalassemia.

Table 1.

The Relevant Clinical, Anthropometric, and Some Laboratory Characteristics of the 56 Patients With β Thalassemia.^a

Parameters	Range	Mean ± SEM
Head circumference, cm	47.00-55.00	50.04 ± 0.29
Postcostal liver size, cm	4.000-10.00	5.893 ± 0.23
Postcostal spleen size—in nonsplenectomized patients, cm	6.0-8.0	7.0 ± 0.47
Splenectomized/nonsplenectomized	24/32	–
Duration on blood transfusion, years	1.60-15.0	7.864 ± 0.50
BMI	11.90-18.40	15.88 ± 0.28
Number of patients on chelation therapy (none/irregular/regular)	12/28/16	–
Hb, g/L	4.000-8.100	5.946 ± 0.14
Ferritin, ng/mL	357.6-3150	2565.8 ± 121.191
pH	7.288-7.523	7.418 ± 0.02
pCO₂, mm Hg	24.90-37.00	31.56 ± 1.15
pO₂, mm Hg	26.00-147.0	94.18 ± 9.87
HCO₃ ⁻, mmol/L	12.80-23.30	20.58 ± 0.66

Abbreviations: Hb, hemoglobin concentration; BMI, body mass index; SEM, standard error of the mean.

^a Data expressed as range and mean ± SEM.

Table 2.

Differences Between Healthy Controls and Pediatric Patients With Thalassemia in the Levels of the Investigated Hypoxia and Oxidative Stress Biomarkers.^a

Parameters	Patients With Thalassemia (56)	Healthy Controls (46)	P Value
Age, years	8.704 ± 0.60	7.91 ± 0.64	–
Gender (male/female)	14 (25.0%)/42 (75.0%)	15 (32.61%)/31 (67.39%)	–
TAC, mmol/L	1.310 ± 0.03	2.259 ± 0.11	<.001
TP, mmol/L	0.054 ± 0.004	0.044 ± 0.001	.069
OSI	4.305 ±0.34	2.163 ± 0.12	<.001
MDA, µmol equivalents/L	1.833 ± 0.09	0.696 ± 0.04	<.001
Lactate, mg/dL	25.41 ± 1.81	14.34 ± 0.91	<.001
Pyruvate, mg/dL	5.429 ± 0.50	5.723 ± 0.67	.254
L/P ratio	7.657 ± 1.04	3.216 ± 0.29	.002
VEGF, pg/mL	354.2 ± 53.69	4.385 ± 0.60	<.001
HIF-1α, pg/mL	89.90 ± 17.72	24.37 ± 2.33	<.001
fDNA, mg/mL	0.170 ± 0.01	0.061 ± 0.01	<.001

Abbreviations: OSI, oxidative stress index; MDA, malondialdehyde; VEGF, vascular endothelial cell growth factor; HIF-1α, hypoxia-induced factor 1α; fDNA, fragmented DNA; TAC, total antioxidant capacity; TP, total peroxide, L/P, lactate/pyruvate; SEM, standard error of the mean.

^a Mann-Whitney U test. Data expressed as mean ± SEM. P ≤ .05 is significant.

Table 3.

The Differences in Some Clinical Characteristics and the Investigated Hypoxia and Oxidative Stress Biomarkers Between the Splenectomized Patients and Nonsplenectomized Patients With Thalassemia.^a

Parameters	Splenectomized Patients (24)	Nonsplenectomized Patients (32)	P Value
Age	11.17 ± 0.73	6.856 ± 0.75	<.001
Gender (male/female)	6/18	8/24	–
Head circumference, cm	50.67 ± 0.37	49.56 ± 0.41	.032
Postcostal liver size, cm	7.583 ± 0.23	4.625 ± 0.13	<.001
Duration on blood transfusion, years	9.925 ± 0.11	6.319 ± 0.56	.001
BMI	15.23 ± 0.44	16.37 ± 0.35	.024
Number of patients on chelation therapy (none/irregular/regular)	2/22/0	10/6/16	–
Hb, g/L	5.983 ± 0.19	5.919 ± 0.10	.791
Ferritin, ng/mL	2705.7 ± 145.73	2461.0 ± 181.53	.947
pH	7.361 ± 0.023	7.440 ± 0.02	.017
pCO₂, mm Hg	33.07 ± 2.11	31.00 ± 1.38	.590
pO₂, mm Hg	52.00 ± 16.44	110.0 ± 9.64	.017
HCO₃ ⁻, mmol/L	19.40 ± 2.09	21.03 ± 0.52	.898
TAC, mmol/L	1.322 ± 0.06	1.401 ± 0.04	.004
TPs, mmol/L	0.048 ± 0.006	0.058 ± 0.004	.119
OSI	4.783 ± 0.52	3.668 ± 0.35	.304
MDA, µmol equivalents/L	2.07 ± 0.13	1.51 ± 0.07	.001
Lactate, mg/dL	26.92 ± 2.90	24.28 ± 2.31	.466
Pyruvate, mg/dL	5.767 ± 0.77	5.175 ± 0.66	.385
L/P ratio	7.400 ± 1.57	7.851 ± 1.42	.824
VEGF, pg/mL	484.5 ± 96.31	256.5 ± 55.41	.012
HIF-1α, pg/mL	55.06 ± 9.25	116.0 ± 29.59	.324
fDNA, mg/mL	0.185 ± 0.02	0.141 ± 0.01	.025

Abbreviations: OSI, oxidative stress index; MDA, malondialdehyde; VEGF, vascular endothelial cell growth factor; HIF-1α, hypoxia-induced factor 1α; fDNA, fragmented DNA; TAC, total antioxidant capacity; TP, total peroxide; L/P, lactate/pyruvate; Hb, hemoglobin concentration.

^aMann-Whitney U test. Data expressed as mean ± SEM, P ≤ .05 is significant.

The percentage of total number of circulating MPs was significantly increased in patients with thalassemia than in the controls. The expression of annexin V (procoagulant MPs) in the patients’ MPs was significantly increased than the controls. The expression of the platelet markers, CD61 and CD41 on MPs (platelet MPs) and platelet activation markers, CD62P were significantly higher on MPs from the patients than on those from the controls. At the same time, the expression of the endothelial marker, CD146 (endothelial MPs) and erythrocyte marker, antiglycophorin A⁺ (erythrocyte MPs) were significantly higher in the patients than in the controls. Microparticles of other cellular origins such as leukocytes (CD45⁺) and monocytes (CD14⁺) were not significantly different between the patients and the controls (Table 4).

Table 4.

Circulating Microparticles in Patients With Thalassemia and Controls.^a

Percentage (%)	Patients With Thalassemia (56)	Healthy Controls (46)	P Value
Total microparticles	62.60 ± 1.70	38.43 ± 2.13	<.001
Annexin V⁺	68.54 ± 1.28	60.43 ± 1.76	<.001
CD61⁺	56.36 ± 1.41	45.35 ± 1.79	<.001
CD41⁺	67.44 ± 1.48	56.48 ± 1.56	<.001
CD62P⁺	29.28 ± 1.48	24.50 ± 1.51	.013
CD146⁺	20.72 ± 1.17	16.85 ± 0.98	.021
CD45⁺	30.78 ± 0.86	28.83 ± 1.02	.170
Antiglycophorin A	15.72 ± 1.62	9.82 ± 0.46	.001
CD14	28.44 ± 1.10	26.55 ± 0.96	.165

Abbreviations: CD, cluster of differentiation; SEM, standard error of the mean.

^a Mann-Whitney U test. Data expressed as mean ± SEM P ≤ .05 is significant.

When comparing splenectomized with nonsplenectomized patients, the percentage of total number of circulating MPs was significantly higher in splenectomized patients than in the nonsplenectomized patients. The MPs of endothelial, platelet, and activated platelet origin were significantly higher in splenectomized patients than the nonsplenectomized patients (Table 5). Many significant correlations were present between the studied parameters (Table 6).

Table 5.

Circulating Microparticles in Splenectomized and Nonsplenectomizd Patients with thalassemi.^a

Percentage (%)	Splenectomized Patients (24)	Nonsplenectomized Patients (32)	P Value
Total microparticles	68.42 ± 2.20	58.31 ± 2.63	.016
Annexin-V⁺	68.25 ± 1.90	68.80 ± 1.81	.673
CD61⁺	59.33 ± 1.95	55.61 ± 1.92	.047
CD41⁺	71.501 ± 2.05	63.69 ± 1.87	.007
CD62P⁺	46.75 ± 1.60	36.23 ± 1.99	.001
CD146⁺	31.87 ± 1.83	25.80 ± 1.26	.012
CD45⁺	31.00 ± 1.25	30.58 ± 1.21	.652
Antiglycophorin A	16.37 ± 2.84	15.11 ± 1.76	.875
CD14	28.04 ± 1.51	28.81 ± 1.63	.807

Abbreviations: CD, cluster of differentiation; SEM, standard error of the mean.

^a Mann-Whitney U test. Data expressed as mean ± SEM P ≤ .05 is significant.

Table 6.

Correlation Results Among Some Investigated Parameters in Patients With Thalassemia.

		L/P Ratio	VEGF	HIF-1α	TAO	OSI	MDA	fDNA	Total MPs	Serum Ferritin
r	L/P ratio	1	0.549	0.492	−0.377	0.328	0.239	0.202	0.351	0.348
P value	L/P ratio		0.001	<0.031	0.183	0.091	0.281	0.285	0.057	0.234
r	VEGF	0.549	1	0.633	−0.477	0.378	0.610	0.379	0.317	0.454
P value	VEGF	0.001		<0.001	0.001	0.015	<0.001	0.038	0.067	0.001
r	HIF-1α	0.492	0.633	1	−0.349	0.401	0.451	0.389	0.511	0.467
P value	HIF-1α	<0.031	0.001		0.146	0.001	0.001	0.001	0.001	0.001
r	TAO	−0.377	−0.477	−0.349	1	−0.525	0.577	−0.369	−0.356	−0.512
P value	TAO	0.183	0.001	0.146		0.006	0.001	0.005	0.007	0.001
r	OSI	0.328	0.378	0.401	−0.525	1	0.463	0.425	0.491	0.377
P value	OSI	0.091	0.015	0.001	0.001		0.001	0.031	0.001	0.041
r	MDA	0.239	0.610	0.451	0.577	0.463	1	0.639	0.519	0.439
P value	MDA	0.281	<0.001	0.001	0.001	0.001		0.001	0.001	0.001
r	fDNA	0.202	0.379	0.389	−0.369	0.425	0. 639	1	0.502	0.410
P value	fDNA	0.285	0.038	0.001	0.005	0.031	0.001		0.003	0.001
r	Total MPs	0.351	0.511	0.317	−0.356	0.491	0.519	0.502	1	0.474
P value	Total MPs	0.057	0.001	0.017	0.007	0.001	0.001	0.003		0.002
r	Serum ferritin	0.348	0.454	0.467	−0.512	0.377	0.439	0.410	0.474	1
P value	Serum ferritin	0.234	0.001	0.001	0.001	0.041	0.001	0.001	0.004

Abbreviations: r, correlation coefficient; OSI, oxidative stress index; MDA, malondialdehyde; VEGF, vascular endothelial cell growth factor; HIF-1α, hypoxia-induced factor 1α; fDNA, fragmented DNA; TAC, total antioxidant capacity; TP, total peroxide; L/P, lactate/pyruvate.

Discussion

Patients with β thalassemia major accumulate body iron over time which causes hepatic, endocrine, and cardiac complications.^27,28 In our patients, the serum level of ferritin was markedly increased. The increased serum ferritin in patients with thalassemia was due to repeated blood transfusion.²⁹ The other potential cause for hyperferritinemia in patients with thalassemia includes the fact that these patients had anoxia due to low hemoglobin level in their blood. Ferritin concentration has been shown to increase in response to stresses such as anoxia.³⁰

In this study, we investigated hypoxia, oxidative stress, DNA damage, and procoagulant biomarkers. Although all these markers have been previously investigated in patients with thalassemia, in our study all these factors are simultaneously analyzed and possible correlations among them are demonstrated. Tissue hypoxia is a common feature of anemia. Hypoxia is accompanied by a significant increase in blood lactate and systemic acidosis as a direct effect of anerobic metabolism. Besides the direct effects of anaerobic metabolism, catecholamine induced stimulation of cellular glycolysis and subsequent synthesis of lactate. In such conditions, accumulated pyruvate is metabolized into lactate. So determination of the L/P ratio provides a more accurate statement of tissue metabolism and the cytosolic redox condition.³¹ In our patients, the increased level of lactate and L/P ratio could be explained by the above-mentioned mechanism.

Under hypoxic/ischemic conditions, HIF induces upregulation of transcription of various cytokines including EPO and VEGF.³² In contrast to the rapid regulation of lactate in response to hypoxia, changes in protein transcription require more time. In patients with thalassemia, the peripheral oxygen demand is greater than the supply for a prolonged period and the resulting tissue hypo oxygenation induces HIF-1-regulated VEGF synthesis.³³

Although high lactate values suggested tissue hypoxia in patients with anemia, it failed to predict the need for RBCs transfusion.^34,35 In addition, this parameter responds quickly to hypoxia, reasons other than anemia, such as hemodynamic alterations, can lead to an elevation in lactate level. In contrast, HIF-regulated changes in VEGF concentration occur relatively slowly. Thus, VEGF is a more appropriate indicator of chronic hypoxic conditions such as anemia.^36,37 The VEGF may serve as an excellent surrogate parameter for identifying tissue hypoxia in patients with anemia and, thus help in determining the need for RBCs transfusion. The marked elevation of VEGF concentrations in our patients with thalassemia suggests insufficient peripheral tissue oxygenation and the need for RBCs transfusion. These data are in accordance with that of the previous studies which showed elevated VEGF concentrations in adult patients with renal³⁶ or sickle cell disease (SCD).³⁷

In splenectomized patients with thalassemia, VEGF level was higher than in nonsplenectomized patients which means more tissue hypoxia in splenectomized patients with thalassemia. This could be due to more severe pulmonary affection of splenectomized patients and pulmonary arterioles occlusion which cause more hypoxia. In accordance with our finding, Butthep et al³⁸ found higher concentration of VEGF in splenectomized patients with thalassemia. Splenectomized patients have higher plasma hemoglobin level than nonsplenectomized patients with thalassemia and higher circulating hemoglobin containing vesicles which worsen the pulmonary hypertension after splenectomy.³⁹ In previous work on our patients with thalassemia, echocardiographic evidence of pulmonary hypertension was detected in 68.75% of the cases. It is more common in splenectomized than nonsplenectomized patients.⁴⁰

In this study, HIF-1α was investigated as a hypoxia biomarker for the first-time in patients with thalassemia. Plasma HIF-1α positively correlated with L/P ratio and VEGF. This indicates the utility of HIF-1α as plasma biomarker of cellular hypoxia in thalassemia which reflects the disease outcomes.

Peroxidative damage of lipids is indicated by the increase in serum MDA levels which is a good indicator of oxidative damage. The extent of lipid peroxidation denotes the amount of free oxygen radicals generated, which have not been scavenged by the defense mechanism. In the present study, MDA in our patients with thalassemia showed a significant increase relative to the control group, indicating an increase in the superoxidation of lipids which results from increased oxidative stress in thalassemia. In accordance with our results, Walter et al⁴¹ found that MDA was significantly increased in patients with thalassemia compared to controls. Cighetti et al⁴² also found elevated level of MDA in thalassemia. Consequently, there is a rationale for iron chelation to eliminate the free iron species, which in this respect, act like antioxidants.⁴³

In the present study, the antioxidant defense was evaluated by measuring TAC in serum. The measurement of different antioxidant molecules separately is labor intensive, time-consuming, and costly. Moreover, some investigators suggest that assessment of TAC of plasma may be more useful than measuring each antioxidants individually since their synergistic interaction could be determined.^44,45 In our patients, the TAC was significantly lower with increased levels of TP and OSI. There are limited studies about the assessment of serum TAC in patients with thalassemia. Ghon et al showed the depletion of antioxidants in patients with thalassemia.⁴⁶ In a study in Italy,⁴⁷ a significant decrease in TAC in patients with thalassemia was reported compared to controls. However, Cakmak et al⁴⁸ reported no significant differences in TAC between thalassemic and control groups, in spite of increased level of oxidant status and OSI. In our splenectomized patients with thalassemia, TAC is markedly reduced and MDA is increased than in nonsplenectomized patients. This finding can be explained by increased hypoxia and oxidative stress present in splenectomized patients. In our patients with thalassemia, there were positive correlations between serum ferritin, VEGF, HIF-1α, MDA, and OSI. All these parameters correlated negatively with TAC. These correlations indicate that more tissue hypoxia in patients with thalassemia induce more iron overload that induces more oxidative stress. In 2 of our females patients, there was a delay in pubertal onset and one of them had cardiomyopathy. In addition, one of our male patient had portal vein thrombosis and 2 had paraparesis. These 5 patients had the highest level of serum ferritin, VEGF, HIF-1α, MDA, OSI, and fDNA, while TAC demonstrated the lowest level.

The elevated level of fDNA in our patients with thalassemia was in accordance with that of Kunwittaya et al⁴⁹ who found increased DNA damage in patients with thalassemia. Also, Söker et al⁵⁰ found elevated DNA damage in patients with thalassemia. Patients with thalassemia have coexisting nutritional deficits⁵¹ and iron-induced oxidative stress,^52,53 both of which are expected to increase DNA damage. In splenectomized patients, fDNA is higher in splenectomized than in nonsplenectomized patients. The fDNA was positively correlated with serum ferritin, MDA, and OSI. Iron overload can stimulate lipid peroxidation, hence generating miscoding DNA adducts in patients with thalassemia and more DNA fragmentation. In accordance with our finding, Meerang et al⁵⁴ found positive correlation between DNA damage with MDA and serum ferritin in patients with thalassemia.

The percentage of total MPs was significantly higher in patients with thalassemia than the controls and higher in splenectomized than in nonsplenectomized patients. Patients who had cardiomyopathy, portal vein thrombosis, or paraparesis had the highest level of total MPs. Patients with β thalassemia have a hypercoagulable state and this hypercoagulabilty is more in splenectomized patients.^55,56 The MPs participate in hemostasis and have procoagulant potential in thalassemia.⁵⁷ The higher expression of annexin V (procoagulant MPs) in our patients means that MPs of patients with thalassemia contain more phosphatidylserine in their outer surfaces.⁵⁸ Such phosphatidylserine may contribute to the pathogenesis of thrombosis.⁵⁹

The elevated endothelial MPs in our patients not only constitute an emerging marker of endothelial dysfunction, but also considered to play a major biological role in inflammation, vascular injury, angiogenesis, and thrombosis. Recent studies indicate that endothelial MPs are able to decrease nitric oxide-dependent vasodilatation, increase arterial stiffness, promote inflammation, and initiate thrombosis.⁶⁰ Platelet MPs supposed to contribute to the development of thrombotic complications in the pathologic states associated with the increase in their blood concentration.⁶¹ Activated platelets MPs provide a procoagulant aminophospholipid surface for the assembly of the specific enzymes of the coagulation cascade. Erythrocyte-derived MPs enhance coagulation activation.⁶² Endothelial MPs, platelet MPs, and activated platelet MPs were significantly higher in our splenectomized patients. Habib et al⁶³ reported that the values of circulating MPs in patients with thalassemia intermedia than the controls with highest statistical significance in MPs of RBCs, endothelial, and leukocytic origins, while platelet MPs were significantly higher in splenectomized patients versus nonsplenectomized patients. Different types of MPs were presents in other types of hemolytic anemia; Wun et al⁶⁴ and van Beers et al⁶⁵ found high numbers of both erythrocyte-derived and platelet-derived MPs in patients with SCD. Nantakomol et al⁶⁶ found increased MPs in individuals with glucose-6-phospate dehydrogenase deficiency; MPs were largely derived from RBCs and platelets. Pattanapanyasat et al⁶⁷ showed that splenectomized patients with β-thalassemia/HbE had significantly higher levels of MPs than nonsplenectomized patients. Shet et al⁶⁸ found endothelial- and monocyte-derived MP in SCD. In our study, we were not able to detect a significant different between patients and controls in the small subset of MPs such as leukocyte or monocyte MPs, this might be due to the differences in the centrifugation forces used to isolate the MPs.

The percentage of total MPs was correlated positively with, serum ferritin, VEGF, OSI, and MDA, suggesting that both hypoxia and oxidative stress are responsible for increasing MPs in thalassemia. During periods of hypoxia, cells are metabolically compromised resulting in cellular dysfunction that can eventually lead to cell death through metabolic failure. Neutrophils bound to the endothelial surface accelerate endothelial cell dysfunction and tissue destruction through a variety of cytotoxic mechanisms. Activated neutrophils mediate a variety of harmful responses, such as production of ROS, cytotoxic enzymes, and cytokines.⁶⁹ The ROS, in large quantities, disrupt the redox balance of cells resulting in oxidative stress and damage to membrane structures possibly leading to membrane MPs release.⁷⁰

Conclusion

The increased tissue hypoxia, oxidative stress in β thalassemia, and its relationship with DNA damage and MPs release could explain many complications of thalassemia and may have therapeutic implications. The VEGF could serve as an important indicator for adequacy of blood transfusion in thalassemia.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Centis

Tabellini

Lucarelli

. The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with β-thalassemia major. Blood. 2000;96(10):3624–3629.

Sansone

Masera

Cantu-Rajnoldi

Terzoli

. An unclassified case of congenital dyserythropoietic anaemia with a severe neonatal onset. Acta Haematol. 1992;88(1):41–45.

Di Matteo

Liuzza

Manicone

. Bone and maxillofacial abnormalities in thalassemia: a review of the literature. J Biol Regul Homeost Agents. 2008;22(4):211–216.

Aizawa

Kohdera

Hiramoto

. Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency. Am J Hematol. 2003;74(1):68–72.

Ivan M, Kondo

Yang

. HIF alpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001;292(5516):464–468.

Jaakola

Mole

Tian

. Targeting of HIFα to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–472.

Bruick

McKnight

. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001;294(5545):1337–1340.

Semenza

GL.

HIF-1, O (2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell. 2001;107(1):1–3.

Young

Hofling

Sands

. VEGF increases engraftment of bone marrow‑derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients. Proc Natl Acad Sci U S A. 2002;99(18):11951–11956.

10.

Zhao

Ishibashi

Hiasa

Tan

Takeshita

Egashira

. Essential role of vascular endothelial growth factor in angiotensin II-induced vascular inflammation and remodeling. Hypertension. 2004;44(3):264–270.

11.

Taher

Isma'eel

Cappellini

. Thalassemia intermedia: revisited. Blood Cells Mol Dis. 2006;37(1):12–20.

12.

Esposito

Breuer

Slotki

Cabantchik

. Labile iron in parenteral iron formulations and its potential for generating plasma non-transferrin-bound iron in dialysis patients. Eur J Clin Invest. 2002:32(suppl 1):42–49.

13.

Gupta

Mishra

Tiwari

In vitro effect of lycopene on oxidative stress in thalassemia major patients. Int J Pharm Sci. 2012;4(3):696–699.

14.

Gelderman

Simak

. Flow cytometric analysis of cell membrane microparticles. Methods Mol Biol. 2008;484:79–93.

15.

Freyssinet

. Cellular microparticles: what are they bad or good for? J Thromb Haemost. 2003;1(7):1655–1662.

16.

Horstman

Jimenez

Ahn

. Endothelial microparticles as markers of endothelial dysfunction (review). Front Biosci. 2004;9:1118–1135.

17.

Wang

Hung

Wang

. Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects. Am J Hypertens. 2007;20(9):957–964.

18.

Helal

Defoort

Robert

. Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: relationship with oxidative stress. Nutr Metab Cardiovasc Dis. 2011;21(9):665–671.

19.

Koracevic

Djordjevic

Andrejevic

Cosic

. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol. 2001;54(5):356–361.

20.

Harma

Erel

. Measurement of the total antioxidant response in preeclampsia with a novel automated method. Euro J Obst Gyn Reprod Biol. 2005;118(1):47–51.

21.

Dawn-Linsley

Ekinci

Ortiz

Rogers

Shea

. Monitoring thiobarbituric acid-reactive substances (TBARs) as an assay for oxidative damage in neuronal cultures and central nervous system. J Neurosci Methods. 2005;141(2):219–222.

22.

Paradones

Illera

Peckham

Stunz

Ashman

. Regulation of apoptosis in vitro in mature spleen T cells. J Immunol. 1993;151(7):3521–3529.

23.

van Beers

Schaap

CLM

Berckmans

. Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematologica. 2009;94(11):1513–1519.

24.

Sommeijer

Joop

Leyte

Reitsma

Ten Cate

. Pravastatin reduces fibrinogen receptor GPIIIa on platelet-derived microparticles in patients with type 2 diabetes. J Thromb Haemost. 2005;3(6):1168–1171.

25.

Trappenburg

van Schilfgaarde

Marchetti

. Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia. Haematologica. 2009;94(7):911–918.

26.

Agouni

Lagrue-Lak-Hal

Ducluzeau

. Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome. Am J Pathol. 2008;173(4):1210–1219.

27.

Lekawanvijit

Chattipakorn

. Iron overload thalassemic cardiomyopathy: iron status assessment and mechanisms of mechanical and electrical disturbance due to iron toxicity. Can J Cardiol. 2009;25(4):213–218.

28.

Galanello

Agus

Campus

Danjou

Giardina

Grady

. Combined iron chelation therapy. Ann N Y Acad Sci. 2010;1202:79–86.

29.

Kennedy

Kohn

Lammi

Clarke

. Iron status and haematological changes in adolescent female in patients with anorexia nervosa. J Paediatr Child Health. 2004;40(8):430–432.

30.

Larade

Storey

. Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in a marine invertebrate. J Exp Biol. 2004;207(pt 8):1353–1560.

31.

Klaus

Heringlake

Gliemroth

Pagel

Staubach

Bahlmann

. Biochemical tissue monitoring

during

hypoxia

and

reoxygenation. Resuscitation. 2003;56(3):299–305.

32.

Becker

Alcasabas

Semenza

Bunton

. Oxygen-independent upregulation of vascular endothelial growth factor and vascular barrier dysfunction during ventilated pulmonary ischemia in isolated ferret lungs. Am J Respir Cell Mol Biol. 2000;22(3):272–279.

33.

Trollmann

Amann

Schoof

. Hypoxia activates the human placental vascular endothelial growth factor system in vitro and in vivo: up-regulation of vascular endothelial growth factor in clinically relevant hypoxic ischemia in birth asphyxia. Am J Obstet Gynecol. 2003;188(2):517–523.

34.

Izraeli

Ben-Sira

Harell

Naor

Ballin

Davidson

. Lactic acid as a predictor for erythrocyte transfusion in healthy preterm infants with anemia of prematurity. J Pediatr. 1993;122(4):629–631.

35.

Frey

Losa

. The value of capillary whole blood lactate for blood transfusion requirements in anaemia of prematurity. Intensive Care Med. 2001;27(1):222–227.

36.

Dunst

Becker

Lautenschläger

. Anemia and elevated systemic levels of vascular endothelial growth factor (VEGF). Strahlenther Onkol. 2002;178(8):436–444.

37.

Solovey

Gui

Ramakrihnan

Steinberg

Hebbel

. Sickle cell anemia as a possible state of enhanced anti-apoptotic tone: survival effect of vascular endothelial growth factor on circulating and unanchored endothelial cells. Blood. 1999;93(11):3824–3830.

38.

Butthep

Rummavas

Wisdpanichkij

Jindadamrongwech

Fucharoen

Bunyaratveg

. Increased circulating endothelial cells, vascular endothelial growth factor and tumor necroses factor in thalassemia. Am J Hematol. 2002;70(2):100–106.

39.

Morris

Kuypers

Kato

. Hemolysis associated pulmonary hypertension in thalassemia. Ann N Y Acad Sci. 2005;1054:481–485.

40.

El-Gezawy

Elsayh

AbdElAziz

NHR

Fathy

Nasif

. Pulmonary hypertension in thalassemic children: does endothelial dysfunction play a role? Alexandria J Ped. 2008;22(2):281–288.

41.

Walter

Fung

Killilea

. Oxidative stress and inflammation in iron-overloaded patients with β-thalassaemia or sickle cell disease. Br J Haematol. 2006;135(2):254–263.

42.

Cighetti

Duca

Bortone

. Oxidative status and malondialdehyde in beta-thalassaemia patients. Eur J Clin Invest. 2002;32(1):55–60.

43.

Fibach

Rachmilewitz

. The role of antioxidants and iron chelators in the treatment of oxidative stress in thalassemia. Ann N Y Acad Sci. 2010;1202:10–16.

44.

Kampa

Nistikaki

Tsaousis

Maliaraki

Notas

Castanas

. A new automated method for the determination of the total antioxidant capacity (TAC) of human plasma, based on the crocin bleaching assay. BMC Clin Pathol. 2002;2(1):3.

45.

Erel

. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical action. Clin Biochem. 2004;37(4);277–285.

46.

Ghone

Kumbar

Suryakar

Katkam

Joshi

. Oxidative stress and disturbance in antioxidant balance in beta thalassemia major. Indian J Clin Biochem. 2008;23(4):337–340.

47.

Hamed

ElMelegy

. Renal functions in pediatric patients with beta-thalassemia major: relation to chelation therapy: original prospective study. Italian J Pediatr. 2010;36:39.

48.

Cakmak

Soker

Koc

Aksoy

. Prolidase activity and oxidative status in patients with thalassemia major. J Clin Lab Anal. 2010;24(1):6–11.

49.

Kunwittaya

Chanarat

Mundee

Mevatee

. Effect of oxidative stress on leukocyte DNA damage in β-thalassemic patients. Siriraj Med J. 2007;59:242–244.www.postersessiononline.com/173580348.../14eha/.../poster_25378.

50.

Söker

Koc

Erel

. Oxidant-antioxidant system and lymphocyte DNA damage in beta thalassemia children. Poster presented at EHA; 2009;14.

51.

Claster

Wood

Noetzli

. Nutritional deficiencies in iron overloaded patients with hemoglobinopathies. Am J Hematol. 2009;84(6):344–348.

52.

Livrea

Tesoriere

Pintaudi

. Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants. Blood. 1996;88(9):3608–3614.

53.

Amer

Ghoti

Rachmilewitz

Koren

Levin

Fibach

. Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants. Br J Haematol. 2006;132(1):108–113.

54.

Meerang

Nair

Sirankapracha

. Accumulation of lipid peroxidation-derived DNA lesions in iron-overloaded thalassemic mouse livers: comparison with levels in the lymphocytes of thalassemia patients. Int J Cancer. 2009;15;125(4):759–766.

55.

Morris

Kuypers

Kato

. Hemolysis associated pulmonary hypertension in thalassemia. Ann N Y Acad Sci. 2005;1054:481–485.

56.

Eldor

Rachmilewitz EA

. The hypercoagulable state in thalassemia. Blood. 2002;99(1):36–43.

57.

Lentz

. Exposure of platelet

membrane phosphatidylserine regulates blood

coagulation. Prog Lipid Res. 2003;42(5):423–438.

58.

Horstman

Jimenez

Bidot

Ahn

. New horizons in the analysis of circulating cell-derived microparticles. Keio J Med. 2004;53(4):210–230.

59.

Mackman

. Role of tissue factor in hemostasis and thrombosis. Blood Cells Mol Dis. 2006;36(2):104–107.

60.

Chironi

Boulanger

Simon

Dignat-George

Freyssinet

Tedgui

. Endothelial microparticles in diseases. Cell Tissue Res. 2009;335(1):143–151.

61.

Sinauridze

Kireev

Popenko

. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost. 2007;97(3):425–434.

62.

Horne

III Cullinane

Merryman

Hoddeson

. The effect of red blood cells on thrombin generation. Br J Haematol. 2006;133(4):403–408.

63.

Habib

Kunzelmann

Shamseddeen

Zobairi

Freyssinet

Taher

. Elevated levels of circulating procoagulant microparticles in patients with beta-thalassemia intermedia. Haematologica. 2008;93(6):941–942.

64.

Wun

Paglieroni

Rangaswami

. Platelet activation in patients with sickle cell disease. Br J Haematol. 1998;100(4):741–749.

65.

van Beers

Schaap

Berckmans

. Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematologica. 2009;94(11):1513–1519.

66.

Nantakomol

Palasuwan

Chaowanathikhom

Soogarun

Imwong

. Red cell and platelet derived microparticles are increased in G6PD deficient subjects. Eur J Haematol. 2012;89(5):423–429.

67.

Pattanapanyasat

Gonwong

Chaichompoo

. Activated platelet-derived microparticles in thalassaemia. Br J Haematol. 2007;136(3):462–471.

68.

Shet

Aras

Gupta

. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood. 2003;102(7):2678–2683.

69.

Inauen

Granger

Meininger

Schelling

Granger

Kvietys

. Anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell injury: role of elastase. Am J Physiol. 1990;259(3 pt 2):925–931.

70.

Semenza

GL.

Cellular and molecular dissection of reperfusion injury: ROS within and without. Circ Res. 2000;86(2):117–118.

Hypoxia Biomarkers,Oxidative Stress,and Circulating Microparticles in Pediatric Patients With Thalassemia in Upper Egypt

Abstract

Keywords

Introduction

Materials and Methods

Patients

Inclusion Criteria

Exclusion Criteria

Biochemical Investigations

Microparticles Isolation and Characterization

Statistical Analysis

Results

Discussion

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

References